Use of akt phosphorylation as a biomarker for prognosing neurodegenerative diseases and treating same

ABSTRACT

The present invention relates to uses of a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, analogues and derivatives thereof, for the treatment of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS). The present invention further provides a method for assessing responsiveness to treatment with the peptide of the invention. In addition, the present invention relates to prognosis of ALS progression, using Akt and phosphorylated Akt as biomarkers.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.14/883,685 filed on Oct. 15, 2015 which is a division of U.S. patentapplication Ser. No. 14/119,891 filed on Nov. 25, 2013, which is aNational Phase of PCT Patent Application No. PCT/IL2012/050185 havingInternational filing date of May 23, 2012, which claims the benefit ofpriority under 35 USC § 119(e) of U.S. Provisional Patent ApplicationNo. 61/488,806 filed on May 23, 2011. The contents of the aboveapplications are all incorporated by reference as if fully set forthherein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 77084SequenceListing.txt, created on Mar. 1,2019 comprising 909 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to uses of a AKT activating agents for thetreatment of neurodegenerative diseases, such as amyotrophic lateralsclerosis (ALS). The present invention relates to uses of a peptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, analogues and derivatives thereof, for thetreatment of neurodegenerative diseases, such as amyotrophic lateralsclerosis (ALS). The present invention further provides a method forassessing responsiveness to treatment with the peptide of the invention.In addition, the present invention relates to prognosis ofneurodegenerative diseases, such as ALS progression, using Akt andphosphorylated Akt as biomarkers.

The serine/threonine protein kinase Akt, also known as protein kinase B(PKB) or RAC-PK, was initially identified as one of the downstreamtargets of phosphatidylinositol-3 kinase (PI3K). Activated Akt plays akey role in mediating signals for cell growth, cell survival(anti-apoptotic), cell-cycle progression, differentiation,transcription, translation and glucose metabolism.

The Akt pathway is damaged in skeletal muscles of amyotrophic lateralsclerosis (ALS) patients, as in SOD1 mice (a mice model for ALS).Although no difference in Akt mRNA levels is found in ALS patients whencompared to control subjects, at the protein level, ALS patients, have asignificantly lower content of the active phosphorylated Akt protein incomparison to healthy control subjects (Leger et al., FASEB J. 2006;20(3):583-585). Akt pathway dysfunction has also been shown inmotoneurons of both sporadic and familial ALS patients (Dewil et al.,Neuropathol Appl Neurobiol., 2007; 33(5):499-509).

The peptides LPPLPYP (SEQ ID NO: 1; also known as Stressin-1) andPYPLPPL (SEQ ID NO: 2, where all residues are in the “D” isomeric form)were first disclosed in WO 2006/021954, where their efficacy inameliorating stress-induced cell death and p53-mediated response wasdemonstrated. According to the disclosure, these peptides are alsouseful in treating inflammatory and autoimmune diseases. Nowhere in thebackground art was it taught or suggested that the activity of peptidescomprising SEQ ID NO: 1 or SEQ ID NO: 2 or derivatives thereof isaffected by the level of Akt and Akt phosphorylation, such that, thetherapeutic effect of these peptides would be significantly higher insubjects having low levels of pAkt and, optionally, low levels ofpAkt:tAkt ratio. In addition, the background art does not teach or evensuggests the use of pAkt and pAkt:tAkt ratio as effective markers forALS and moreover for staging the progression of ALS in ALS patients.

ALS is a devastating and rapidly fatal disease with currently only oneavailable, FDA-approved, modestly effective treatment. The approvetherapy, Rilutek® (Riluzole), has a modest benefit estimated to be athree month extension in patient survival.

There is therefore an urgent need for new therapies. Recent attempts tofind molecules that could provide a beneficial therapy for ALS includethe finding of1,4-Diaza-bicyclo[3.2.2]non-6-en-4-yl)-heterocyclyl-methanone ligandsdisclosed in US Patent Application, Publication No. 2010/0298306. Theligands are directed to treatment of any Nicotinic AcetylcholineReceptors, inter alia, ALS. In addition, US Patent Application,Publication No. 2010/0099700, discloses use of hydrogenatedpyrido(4,3-b)indole for treating ALS. US Patent Application, PublicationNo. 2009/0324549 discloses methods for treating ALS comprisingadministration to a patient in need thereof, proteins and/or peptidescharacterized in that they originate from the gene which results fromthe retention of the intron 3 of the gene SMN (or survival motor neuron)identified in the gene bank with the access number AY876898.

WO 2011/017030 discloses a method of treating a disease associated withexcess activation of monocytes to activated macrophages, including,inter alia, ALS said method comprising administering atherapeutically-effective amount of an oxidative agent to a subject inneed thereof, wherein said oxidative agent is selected from the groupconsisting of non-halogen activated-oxygen compounds, non-oxygenactivated-halogen compounds, and N-halo compounds. According to WO2011/017030 the non-halogen activated-oxygen compounds are selected frompotassium nitrate (KNO₃), permanganate salts, ammonium cerium(IV)nitrate, hexavalent chromium compounds, chromate/dichromate compounds,ammonium silver nitrate, sulfoxides, persulfuric acid, osmium tetroxide(OsO₄), nitric acid, nitrous oxide (N₂O), hydrogen peroxide, organicperoxides, superoxides, and ozone; the non-oxygen activated-halogencompounds are selected from fluorine, chlorine, bromine, and iodine; theN-halo compounds are selected from the group consisting ofN-halophthalimide, N-halosuccinimide, N-halosaccharin,N,N-dihalourethane, N-haloacetanilide, 1,3-dihalo-5,5-dimethylhydantoin,trihaloisocyanuric acid and sodium dihaloisocyanurate; and the oxidativeagent is selected from 1,3-dichloro-5,5-dimethylhydantoin andchloramine-T.

The development of the first genetically based mouse model of ALS in1994, energized the field of preclinical testing despite numerousunforeseen complexities along the way. Transgenic mutant SOD1 mice, theonly ALS mouse models currently available, have mutations in the Cu/ZnSuperoxide Dismutase 1 gene (SOD1) which account for ˜20% of FamilialALS (FALS) cases, corresponding to 2-3% of all ALS cases. Transgenicmutant SOD1 mice exhibit all of the histopathological hallmarks observedclinically in sporadic and familial ALS.

Because there is no obvious mutational hotspot and no clear correlationbetween the level of enzymatic activity of the mutant SOD1 protein andthe observed disease phenotype or clinical progression, SOD1 is thoughtto act primarily via a toxic gain of function in ALS, although loss offunction may also contribute to disease pathophysiology. It is generallythought that the different mutant SOD1 proteins are likely to cause ALSby a similar mechanism.

Several transgenic mouse models have been generated to model mutationsfound in FALS patients. In all of these mouse models, massive death ofmotor neurons in the ventral horn of the spinal cord and loss ofmyelinated axons in ventral motor roots ultimately leads to paralysisand muscle atrophy. All of these mouse models have been reported toexhibit the same histopathological hallmarks associated with ALS inhumans: progressive accumulation of detergent-resistant aggregatescontaining SOD1 and ubiquitin and aberrant neurofilament accumulationsin degenerating motor neurons. In addition to neuronal degeneration,reactive astroglia and microglia have also been detected in diseasedtissue in the mice, similar to that observed in humans.

Despite these histopathological similarities, the timing of onset andrate of disease progression differ (often dramatically) among thevarious SOD1 transgenic mouse models.

ALS is commonly assessed by neurological score and weight loss. As usedherein, the term “neurological score” and “neurologic score” areinterchangeably used herein to describe the common standards forassessing the presence or stage of a neurologic disease, such as ALS.Some example of commonly used neurological scoring systems include:measurements of splay (or other measures of paralysis) and beam walk.

In SOD1 mice, regardless of which neurological scoring system is used,scores are typically assessed for both hind legs of the SOD1 mice. Theexample neurological scoring system below employs a scale of zero tofour(www(dot)researchals(dot)org/uploaded_files/p41_jax_sod1manual_20091202_29aPcx(dot)pdf).Example of score criteria used to assign each score under this systemare as follows:

-   -   Score of 0: full extension of hind legs away from lateral        midline when mouse is suspended by its tail, and mouse can hold        this for two seconds, suspended two to three times.    -   Score of 1: collapse or partial collapse of leg extension        towards lateral midline (weakness) or trembling of hind legs        during tail suspension.    -   Score of 2: toes curl under at least twice during walking of 12        inches, or any part of foot is dragging along cage bottom/table.    -   Score of 3: rigid paralysis or minimal joint movement, foot not        being used for generating forward motion.    -   Score of 4: mouse cannot right itself within 30 seconds after        being placed on either side.

U.S. Pat. No. 7,659,243 discloses the use of angiogenin, or a fragmentor variant thereof, to treat diseases characterized by neuronal injuryor death, or axonal degeneration, especially neurodegenerative diseasessuch as ALS. According to the disclosure, the neuroprotective effect ofangiogenin involves the activation of the PI3K/Akt pathway.

U.S. Pat. Nos. 7,030,090 and 7,517,857 disclose a peptide thatstimulates Akt phosphorylation via activation of formyl peptide receptoror formyl peptide receptor-like 1, the peptide comprises an amino acidsequence of WX1X2MX3X4, where X1=K, R, E, H or D, X2=G, Y, H, E or W,X3=V or G and X4=D-Me or G.

U.S. Pat. No. 7,622,455 discloses a method of treating ALS comprisingadministering to the cerebrospinal fluid of a subject in need thereofantisense oligonucleotides complementary to SOD1 nucleic acids.

There is an unmet need for novel methods for slowing down the rate ofprogression of neurodegenerative diseases, such as ALS, assessingresponsiveness to treatment of the disease, and staging the disease in amanner that is specific, safe and effective.

SUMMARY OF THE INVENTION

The present invention provides methods for treating neurodegenerativediseases, such as ALS and variants of this disease, the method comprisesassessing pAkt level and the level of ratio pAkt to total Akt (tAkt) ina subject in need thereof, followed by administering to a subject havingpAkt and/or pAkt:tAkt ratio significantly below a predeterminedthreshold level, a composition comprising a peptide having an amino acidsequence comprising a sequence selected from the group consisting of SEQID NO: 1 and SEQ ID NO: 2.

While use of the peptides of the invention for amelioratingstress-induced cell death and p53-mediated response has been described,the present invention demonstrates that said peptides are useful inincreasing the levels of Akt phosphorylation, thereby better definingthe population that would benefit from treatment with the peptide.

It is now disclosed for the first time that a peptide having an aminoacid sequence consisting of SEQ ID NO: 1 or SEQ ID NO:2 activates theAkt pathway. The peptide-induced activation of the Akt pathway in aspecific manner was detected shortly after introducing said peptide tomacrophages culture. The peptide's induced activation of the Akt pathwaywas also observed in vivo. Thereby, the invention demonstrates that thepeptides of the invention are useful for treating diseases associatedwith low Akt phosphorylation (pAkt) or low pAkt:tAkt ratio.

Surprisingly, administering the peptide of the invention to SOD1 miceenhanced survival and prolonged the life span of said mice in asignificant manner (10 days longer; P=0.008) in comparison tonon-treated mice. In addition to extended survival, the peptide of theinvention significantly delays disease progression by delaying theappearance of the disease symptoms, primarily, the challenged mobility.

Without wishing to be bound by theory or mechanism, the advantages ofthe peptide of the invention over other treatments of ALS known to datemay be attributed to the fact that the peptide was tested, and shown tobe effective not only before disease onset but also after disease onsetincluding at very late stage of disease.

Advantageously, use of the peptide of the invention is relatively safe,as administration of the peptide in a dose that is four times higherthan the effective dose, daily, for 28 days, did not initiate anydetectable side effect in healthy mice. Accordingly, the peptide of theinvention is suitable for chronic use. In addition, the peptide of theinvention was shown effective when administered intravenously (IV),intraperitoneally (IP) or in combination of these two modes of delivery.

The present invention also highlights the use of Akt phosphorylation andpAkt:tAkt ratio as biomarkers that are significantly useful for stagingand monitoring the progression of ALS. Based on the teaching of thepresent invention, Akt phosphorylation and pAkt:tAkt ratio distinguishbetween fast progression to low progression of ALS. Specifically, lowAkt phosphorylation and, optionally, low pAkt:tAkt ratio indicaterapidly progressing ALS whereas high levels of Akt phosphorylation and,optionally, high pAkt:tAkt ratio indicate slow progression of thedisease. The biomarkers of the invention are also useful forestablishing responsiveness to treatment with the peptides of theinvention. It is shown that high levels of pAkt and high levels ofpAkt:tAkt ratio (e.g. above 1) correlate with responsiveness totreatment of ALS with the peptides of the invention.

According to an aspect of some embodiments of the present inventionthere is provided a method for prognosticating the progression of aneurodegenerative disease in a subject, comprising (a) assessing thevalue of at least one marker selected from: pAkt and pAkt:tAkt ratio, ina bodily sample derived from the subject; and (b) obtaining the ratiobetween the value of the marker and the value of the marker in a controlsample, wherein a level of pAkt or pAkt:tAkt ratio significantly below acontrol value indicates a rapid disease.

According to an aspect of some embodiments of the present inventionthere is provided a method for treating a neurodegenerative disease in asubject in need thereof, comprising administering to a subject having arapidly progressing neurodegenerative disease a therapeuticallyeffective amount of an agent capable of activating a Akt pathway,thereby treating the neurodegenerative disease.

According to an aspect of some embodiments of the present inventionthere is provided a method for treating a neurodegenerative disease in asubject in need thereof, comprising (a) assessing the level of pAkt andpAkt:tAkt ratio in a bodily sample derived from the subject; and (b)administering a therapeutically effective amount of a peptide comprisingan amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2 and an analog or a derivative thereof, to a subjecthaving pAkt level or pAkt:tAkt ratio significantly below a controlvalue.

According to an aspect of some embodiments of the present inventionthere is provided a method for treating amyotrophic lateral sclerosis(ALS) in a subject in need thereof, comprising (a) assessing the levelof pAkt and pAkt:tAkt ratio in bodily sample derived from the subject;and (b) administering a therapeutically effective amount of a peptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2 and an analog or a derivative thereof, to asubject having pAkt level or pAkt:tAkt ratio significantly below acontrol value.

According to an aspect of some embodiments of the present inventionthere is provided a method for assessing responsiveness to treatment ofa disease with a peptide comprising an amino acid sequence of SEQ ID NO:1, SEQ ID NO: 2 or an analog or a derivative thereof, or apharmaceutical composition comprising same, the method comprising:assessing the level of pAkt or pAkt:tAkt ratio in bodily sample derivedfrom a subject, wherein responsiveness to treatment is indicated by apAkt level or pAkt:tAkt ratio significantly above a value of a pAktlevel or pAkt:tAkt ratio in the subject prior to the treatment.

According to an aspect of some embodiments of the present inventionthere is provided a kit for diagnosing a neurodegenerative disease orprognosticating its progression in a subject comprising i) means forcollecting a bodily sample from a subject and ii) means for determiningthe level of pAkt and tAkt in the sample.

According to an aspect of some embodiments of the present inventionthere is provided a method of determining the efficacy of treatment of aneurodegenerative disease in a subject in need thereof comprisingdetermining in a sample from the subject the effect of a peptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2 on the level of pAkt and pAkt:tAkt ratio,wherein an increase in the level indicates that the subject isresponsive to treatment with the peptide.

According to some embodiments of the invention, the disease is selectedfrom the group consisting of: amyotrophic lateral sclerosis (ALS),primary lateral sclerosis (PLS) and spinal muscular atrophy (SMA).

According to some embodiments of the invention, the disease isamyotrophic lateral sclerosis (ALS).

According to some embodiments of the invention, the disease is selectedfrom the group consisting of: Alzheimer's disease, Parkinson's disease,glaucoma, macular degeneration, hypoxia, fulminant toxic liver, kidneyfailure and infertility.

According to some embodiments of the invention, the bodily sample isselected from the group consisting of: muscle, blood, blood plasma,lymph fluid, lymphocytes and leukocytes.

According to some embodiments of the invention, the control valuecorresponds to pAkt level or pAkt:tAkt ratio in a sample selected fromthe group consisting of: a bodily sample of a healthy individual, abodily sample of an individual not afflicted with any neurodegenerativedisease, a bodily sample of an individual afflicted with a slowlyprogressing neurodegenerative disease and a sample derived from an ALSsubject having a slow disease.

According to some embodiments of the invention, the agent capable ofactivating the Akt pathway comprises a peptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2 and an analog or a derivative thereof.

According to some embodiments of the invention, the agent capable ofactivating the Akt pathway is selected from the group consisting of ainsulin-like growth factor 1 (IGF-I), vascular endothelial growth factor(VEGF), angiogenin, naphtho[1,2-b]furan-4,5-dione (NFD), ciliaryneurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF),growth factor-beta (TGF-beta), glial-cell-line-derived neurotrophicfactor (GDNF), Hepatic growth factor (HGF), fibroblast growth factor(FGF) and epidermal growth factor (EGF).

According to some embodiments of the invention, the agent capable ofactivating the Akt pathway is administered by a route of administrationselected from the group consisting of: oral, transdermal, parenteral,transmucosal, intrathecal, intracerebroventricular (ICV), intranasal,sublingual, intravenous and intraperitoneal.

According to some embodiments of the invention, determining aprogression of the neurodegenerative disease is effected according tothe method of the present invention.

According to some embodiments of the invention, the bodily sample isderived from the peripheral blood, the lymph system or a muscle of thesubject.

According to some embodiments of the invention, the peripheral blood orthe lymph system comprises lymphocytes.

According to some embodiments of the invention, the peptide is comprisedin a pharmaceutical composition in combination with at least one moretherapeutic drug.

According to some embodiments of the invention, the at least one moretherapeutic drug is selected from the group consisting of: an oxidativeagent, non-halogen activated-oxygen compounds, non-oxygenactivated-halogen compounds, N-halo compounds and riluzole.

According to some embodiments of the invention, the pharmaceuticalcomposition further comprises a pharmaceutical acceptable excipient,carrier or diluent.

According to some embodiments of the invention, treating comprisesattenuating the progression of the disease, alleviating symptoms of thedisease, delaying the appearance of disease symptoms or improvingmanagement of the disease.

According to some embodiments of the invention, the administering thepeptide by a route of administration selected from the group consistingof: oral, transdermal, parenteral, transmucosal, intrathecal,intracerebroventricular (ICV), intranasal, sublingual, intravenous andintraperitoneal.

According to some embodiments of the invention, the peptide consists ofan amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2 and ananalog or a derivative thereof.

According to some embodiments of the invention, the bodily sample isderived from the peripheral blood, the lymph system or the muscle of thesubject.

According to some embodiments of the invention, the peripheral blood orthe lymph system comprises lymphocytes.

According to some embodiments of the invention, treating comprisesadministering the peptide by a route of administration selected from thegroup consisting of: oral, transdermal, parenteral, transmucosal,intrathecal, intracerebroventricular (ICV), intranasal, sublingual,intravenous and intraperitoneal.

According to some embodiments of the invention, the kit furthercomprises a standard, a calibration curve or an index indicating acontrol value of the pAkt and the tAkt.

According to some embodiments of the invention, the control valuecorresponds to pAkt level or pAkt:tAkt ratio in a sample selected fromthe group consisting of: a bodily sample of a healthy individual, abodily sample of an individual not afflicted with any neurodegenerativedisease, a bodily sample of an individual afflicted with a slowlyprogressing neurodegenerative disease and a sample derived from asubject having a slow disease.

According to some embodiments of the invention, the means fordetermining the levels of pAkt comprise at least one antibody directedto pAkt.

According to some embodiments of the invention, the means fordetermining the levels of tAkt comprise at least one antibody directedto tAkt.

These and other embodiments of the present invention will becomeapparent in conjunction with the figures, description and claims thatfollow.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1A presents Akt phosphorylation in macrophages treated with apeptide having the amino acid sequence of SEQ ID NO: 1.

FIG. 1B shows Akt phosphorylation in mice treated with the peptide ofSEQ ID NO: 1 as compared to non-treated mice.

FIG. 2 shows survival curve after disease onset (week 15 onwards) oftreated (light squares) and untreated (control; dark squares) SOD1 micetogether with the extent of significance, as evaluated by Chi test (*for P=<0.05; ** for P=<0.01 and *** for P=<0.001). Mice were treatedwith the peptide of the invention starting at disease onset as indicatedby weigh loss (average—90 days, 400 μg 200 IP+200 IV) and were comparedto control (n=21+21) mice (untreated or treated with PBS).

FIG. 3 exhibits the average neurologic disability score in treated(light squares) and untreated (control; dark squares) SOD1 mice togetherwith the extent of significance, as evaluated by Student's T test (* forP=<0.05; ** for P=<0.01 and *** for P=<0.001). Mice were treated withthe peptide of the invention starting at disease onset (average 90 days,400 μg 200 IP+200 IV) and were compared to control (n=21+21) mice(untreated or treated with PBS).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention provides methods for treating diseases associatedwith low Akt phosphorylation levels, such as ALS, comprisingadministering to a subject in need thereof a pharmaceutical compositioncomprising a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, analogues or derivativesthereof. The present invention also provides methods for assessingresponsiveness to treatment of diseases associated with low Aktphosphorylation levels, such as ALS with the peptide of the invention,comprising evaluating the level of Akt phosphorylation and pAkt:tAktratio prior to treatment, and comparing the values to a control value,thereby determining responsiveness to therapy with the peptide of theinvention.

According to one embodiment, there is provided a method for treating aneurodegenerative disease in a subject in need thereof, comprisingadministering to a subject having a rapidly progressingneurodegenerative disease a therapeutically effective amount of an agentcapable of activating a Akt pathway, thereby treating theneurodegenerative disease.

According to one embodiment, there is provided a method for treating aneurodegenerative disease in a subject in need thereof, comprising (a)assessing the level of pAkt and pAkt:tAkt ratio in a bodily samplederived from the subject; and (b) administering a therapeuticallyeffective amount of a peptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and an analog ora derivative thereof, to a subject having pAkt level or pAkt:tAkt ratiosignificantly below a control value.

The term “treating” as used herein includes prophylactic and therapeuticuses, and refers to the alleviation of symptoms of a particular diseasein a patient, and/or improvement of an ascertainable measurementassociated with a particular disorder. More specifically, in the contextof this invention “treating a disease associated with low levels of Aktphosphorylation”, particularly ALS, means attenuating the progression ofsaid disease and/or alleviating symptoms of said disease and/orimproving management of said disease. This modulation can be measured byassessing the level of Akt phosphorylation upon treatment and prior totreatment, where assessment of Akt phosphorylation may be carried out inways which are routine in the art, for example, mass spectroscopy.

As used herein the term “subject” refers to a mammalian subject, e.g.human subject, who is at risk of developing a neurodegenerative diseaseor who exhibits clinical signs of a neurodegenerative disease. Thesubject may be of any age and gender.

Examples of neurodegenerative diseases which may be treated according tothe present invention include, but are not limited to, AmyotrophicLateral Sclerosis (ALS), primary lateral sclerosis (PLS), spinalmuscular atrophy (SMA), Alzheimer's disease, Parkinson's disease,glaucoma, macular degeneration, hypoxia, fulminant toxic liver, kidneyfailure, infertility, type 1 diabetes, multiple sclerosis, systemiclupus erythematosis, autoimmune uveitis, graft versus host disease,graft rejection, arthritis, systemic inflammatory response syndrome(SIRS), inflammatory bowel disease (IBD), adult respiratory distresssyndrome (ARDS), psoriasis, atherosclerosis.

According to a specific embodiment, the neurodegenerative disease isALS.

As used herein “amyotrophic lateral sclerosis (ALS)” also referred to as“Lou Gehrig's disease” refers to a progressive, fatal, neurodegenerativedisease caused by the degeneration of motor neurons, the nerve cells inthe central nervous system that control voluntary muscle movement. Theterm ALS includes sporadic and familial ALS, ALS at any rate ofprogression (i.e. rapid or slow progression) and ALS at any stage (e.g.prior to onset, at onset and late stages of ALS).

ALS typically causes muscle weakness and atrophy throughout the body asboth the upper and lower motor neurons degenerate, ceasing to sendmessages to muscles. Unable to function, the muscles gradually weaken,develop fasciculations (twitches) because of denervation, and eventuallyatrophy because of that denervation. Affected subjects may ultimatelylose the ability to initiate and control all voluntary movement; bladderand bowel sphincters and the muscles responsible for eye movement areusually, but not always, spared.

Cognitive function is generally spared except in certain situations suchas when ALS is associated with frontotemporal dementia. However, thereare reports of more subtle cognitive changes of the frontotemporal typein many patients when detailed neuropsychological testing is employed.Sensory nerves and the autonomic nervous system, which controlsfunctions like sweating, generally remain functional. ALS as used hereinrefers to all the above exemplary manifestations. ALS, as used hereinrefers to hereditary and sporadic ALS.

Both hereditary causes and environmental risks may contribute to onsetof disease. For instance, an inherited genetic defect on chromosome 21(coding for superoxide dismutase) is associated with approximately 20%of familial cases of ALS. This mutation is believed to be autosomaldominant. The most common ALS causing SOD1 mutation in North America isA4V, characterized by an exceptionally rapid progression from onset todeath. The children of those diagnosed with familial ALS have a higherrisk factor for developing the disease; however, those who have closefamily members diagnosed with sporadic ALS have no greater a risk factorthan the general population, suggesting an environmental or othernon-genetic cause.

Furthermore, environmental causative factors have been suggested for theincreased incidence of ALS. These include, prolonged exposure to adietary neurotoxin called BMAA produced by cyanobacteria which is one ofseveral possible neurotoxic compounds found in the seed of the cycadCycas circinalis, a tropical plant found in Guam; Exposure topesticides; toxic exposure such as nerve gas.

As mentioned above, the method of the invention is directed, inter alia,for treating ALS. The treatment may be initiated at any stage of thedisease, including following detection of ALS symptoms.

Detection of ALS may be determined by the appearance of differentsymptoms depending on which motor neurons in the body are damaged first(and consequently which muscles in the body are damaged first). Ingeneral, ALS symptoms include the earliest symptoms which are typicallyobvious weakness and/or muscle atrophy. Other symptoms include musclefasciculation (twitching), cramping, or stiffness of affected muscles,muscle weakness affecting an arm or a leg and/or slurred and nasalspeech. Most ALS patients experience first symptoms in the arms or legs.Others first notice difficulty in speaking clearly or swallowing. Othersymptoms include difficulty swallowing, and loss of tongue mobility. Asmall proportion of patients experience respiratory difficulties.

The symptoms may be also classified by the part of neuronal system thatis degenerated, namely, upper motor neurons and lower motor neurons.Symptoms of upper motor neuron degeneration include tight and stiffmuscles (spasticity) and exaggerated reflexes (hyperreflexia) includingan overactive gag reflex. Symptoms of lower motor neuron degenerationinclude muscle weakness and atrophy, muscle cramps, and fleetingtwitches of muscles that can be seen under the skin (fasciculations). Tobe diagnosed with ALS, patients must have signs and symptoms of bothupper and lower motor neuron damage that cannot be attributed to othercauses.

Alternatively, treatment may be initiated at progressive stages of thedisease, e.g. when muscle weakness and atrophy spread to different partsof the body and the subject has increasing problems with moving [e.g.the subject may suffer from tight and stiff muscles (spasticity), fromexaggerated reflexes (hyperreflexia), from muscle weakness and atrophy,from muscle cramps, and/or from fleeting twitches of muscles that can beseen under the skin (fasciculations)], swallowing (dysphagia), speakingor forming words (dysarthria).

The present invention further provides a method for treating amyotrophiclateral sclerosis (ALS) in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2 and an analog or aderivative thereof, thereby treating the ALS.

The present invention contemplates treatment using the peptidecomprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or ananalog or a derivative thereof.

The present invention encompasses any analog, derivative, and conjugatecontaining the peptides of the invention, the amino acid sequence ofwhich is shown herein so long as the peptide is capable of inducing Aktphosphorylation. Thus, the present invention encompasses peptidescontaining non-natural amino acid derivatives or non-protein sidechains.

The term “analog” includes any peptide or polypeptide having an aminoacid sequence substantially identical to one of the sequencesspecifically shown herein in which one or more residues have beenconservatively substituted with a functionally similar residue and whichdisplays the abilities as described herein. Examples of conservativesubstitutions include the substitution of one non-polar (hydrophobic)residue such as isoleucine, valine, leucine or methionine for another,the substitution of one polar (hydrophilic) residue for another such asbetween arginine and lysine, between glutamine and asparagine, betweenglycine and serine, the substitution of one basic residue such aslysine, arginine or histidine for another, or the substitution of oneacidic residue, such as aspartic acid or glutamic acid for another.

A peptide derivative refers to a molecule comprising the amino acidsequence of a peptide of the invention subject to various changes,including, but not limited to, chemical modifications, substitutions,insertions, extensions and deletions where such changes do not destroythe anti-inflammatory or anti-apoptotic activity of the peptide, andsuch derivative is not a known peptide or protein. “Peptide derivative”is intended to include peptide mimetics, as described hereinbelow.

Peptide derivatives having chemical modifications include, for example,peptides having one or more residues chemically derivatized by reactionof side chains or functional groups. Such derivatized molecules include,for example, those molecules in which free amino groups have beenderivatized to form amine hydrochlorides, p-toluene sulfonyl groups,carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups orformyl groups. Free carboxyl groups may be derivatized to form salts,methyl and ethyl esters or other types of esters or hydrazides. Freehydroxyl groups may be derivatized to form O-acyl or O-alkylderivatives. The imidazole nitrogen of histidine may be derivatized toform N-im-benzylhistidine. Also included as chemical derivatives arethose peptides, which contain one or more naturally occurring amino acidderivatives of the twenty standard amino acid residues. For example:4-hydroxyproline may be substituted for proline; 5-hydroxylysine may besubstituted for lysine; 3-methylhistidine may be substituted forhistidine; homoserine may be substituted or serine; and ornithine may besubstituted for lysine.

In addition, a peptide derivative can differ from the natural sequenceof the peptides of the invention by chemical modifications including,but are not limited to, terminal-NH₂ acylation, acetylation, orthioglycolic acid amidation, and by terminal-carboxyl-amidation, e.g.,with ammonia, methylamine, and the like.

Preferred peptide derivatives are retro-inverso peptides. In particularembodiment the methods of the invention provides uses of a peptidehaving the amino acid sequence set forth in SEQ ID NO: 1 or aretro-inverso derivative thereof as set forth in SEQ ID NO: 2.

As used herein, the term “retro-inverso peptide” of the peptide of SEQID NO: 1, for example, as used in a variation of the invention, isintended to encompass peptides in which the sequence of the amino acidsis reversed as compared to the sequence in SEQ ID NO: 1 and consist ofD-amino acids in reversed order. Retro-inverso peptides consist ofD-amino acids in reversed order, resulting in an altered peptidebackbone but unchanged orientation of the side chains. Retro-inversopeptides are usually advantageous over the original peptide as they areresistant to proteases.

Peptides of the present invention also include any peptide having one ormore additions and/or deletions of residues relative to the sequence ofthe peptides of the invention, the sequence of which are shown herein,so long as the requisite induction activity of Akt phosphorylation ismaintained.

Addition of amino acid residues may be performed at either terminus ofthe peptides of the invention for the purpose of providing a “linker” bywhich the peptides of this invention can be conveniently bound to acarrier. Such linkers are usually of at least one amino acid residue andcan be of 40 or more residues, more often of 1 to 10 residues. Typicalamino acid residues used for linking are tyrosine, cysteine, lysine,glutamic and aspartic acid, or the like.

A peptide of the invention may also be conjugated to itself oraggregated in such a way as to produce a large complex containing thepeptide. Such large complex may be advantageous because it has newbiological properties such as longer half-life in circulation or greateractivity.

Peptidomimetics are small molecules that can bind to proteins bymimicking certain structural aspects of peptides and proteins. They areused extensively in science and medicine as agonists and antagonists ofprotein and peptide ligands of cellular and other receptors, and assubstrates and substrate analogs for enzymes.

A primary goal in the design of peptide mimetics has been to reduce thesusceptibility of mimics to cleavage and inactivation by peptidases. Inone approach, one or more amide bonds have been replaced in anessentially isosteric manner by a variety of chemical functional groups,including, but not limited to urea bond, carbamate bond, sulfonamidebond, hydrazine bond, or any other covalent bond. In another approach, avariety of uncoded or modified amino acids such as D-amino acids andN-methyl amino acids have been used to modify mammalian peptides.

As mentioned, treating the neurodegenerative disease may be affected byadministration of a therapeutically effective amount of an agent capableof activating a Akt pathway.

As used herein, the phrase “agent capable of activating a Akt pathway”refers to a molecule that upregulates phosphorylation of Akt per se or adown-stream signaling effector thereof (i.e., indirect activation).

Any agent capable of activating a Akt pathway may be used in accordancewith the present teachings. Exemplary agents which may used include, butare not limited to, insulin-like growth factor 1 (IGF-I), vascularendothelial growth factor (VEGF), angiogenin,naphtho[1,2-b]furan-4,5-dione (NFD), ciliary neurotrophic factor (CNTF),brain-derived neurotrophic factor (BDNF), growth factor-beta (TGF-beta),glial-cell-line-derived neurotrophic factor (GDNF), Hepatic growthfactor (HGF), fibroblast growth factor (FGF) and epidermal growth factor(EGF).

Each of the agents capable of activating a Akt pathway or the peptidecomprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or ananalog or a derivative thereof as described hereinabove can beadministered to the subject per se or as part of a pharmaceuticalcomposition.

As used herein a “pharmaceutical composition” refers to a preparationcomprising the peptide of the invention, with other chemical componentssuch as physiologically suitable carriers and excipients. The purpose ofa pharmaceutical composition is to facilitate administration of acompound to a patient in need thereof.

The composition of the invention may be administered by any conventionaland appropriate route of administration, including, but not limited to,oral, intravenous, intramuscular, subcutaneous, intrathecal, topical,rectal, buccal, inhalational, intranasal transdermal, parenteral,transmucosal, sublingual, intravenous and intraperitoneal.

Hereinafter, the term “oral administration” includes, but is not limitedto, administration by mouth for absorption through the gastrointestinaltract (peroral) wherein the drug is swallowed, or for trans-mucosalabsorption in the oral cavity by buccal, gingival, lingual, sublingualand oro-pharyngeal administration. Compositions for oral administrationinclude powders or granules, suspensions or solutions in water ornon-aqueous media, sachets, capsules or tablets. The oral compositioncan optionally contain inert pharmaceutical excipients such asthickeners, diluents, flavorings, dispersing aids, emulsifiers, binders,preservatives and the like.

The term “parenteral administration” as used herein indicates any routeof administration other than via oral administration and includes, butis not limited to, administration by intravenous drip (IV) or bolusinjection, intraperitoneal (IP), intrathecal, subcutaneous, or intramuscular injection, topical, transdermal, rectal, intranasal (IN)administration or by inhalation.

According to one embodiment, the peptide as set forth in SEQ ID NO: 1,SEQ ID NO: 2 and an analog or a derivative thereof is administered via acombination of two routes (e.g. intraperitoneal and intravenous routes).

According to one embodiment, the peptide of the invention isadministered orally.

Formulations for parenteral administration include but are not limitedto sterile aqueous solutions which can also contain buffers, diluentsand other suitable additives.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants for exampleDMSO, or polyethylene glycol are generally known in the art.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers.

In soft capsules, the active compounds may be dissolved or suspended insuitable liquids, such as fatty oils, liquid paraffin, or liquidpolyethylene glycols. In addition, stabilizers may be added. Allformulations for oral administration should be in dosages suitable forthe chosen route of administration.

Alternatively, the compounds of the present invention can beincorporated into oral liquid preparations such as aqueous or oilysuspensions, solutions, emulsions, syrups, or elixirs, for example.Moreover, formulations containing these compounds can be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can contain conventional additives, likesuspending agents, such as sorbitol syrup, methyl cellulose,glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and hydrogenated edible fats;emulsifying agents, such as lecithin, sorbitan monooleate, or acacia;nonaqueous vehicles (which can include edible oils), such as almond oil,fractionated coconut oil, oily esters, propylene glycol, and ethylalcohol; and preservatives, such as methyl or propyl p-hydroxybenzoateand sorbic acid.

For administration by inhalation, the peptides for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from a pressurized pack or a nebulizer with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the peptide and a suitable powder base suchas lactose or starch.

The pharmaceutical compositions of the invention are also useful fortopical application. As used herein, the term “topical” means“pertaining to a particular surface area”, e.g. skin and mucosa, and thetopical agent applied to a certain area of said surface will affect onlythe area to which it is applied. The formulations of thepeptides/peptide analogs may be administered topically as a gel,ointment, cream, emulsion, sustained release formulation including atransdermal patch, and may comprise liposomes and any otherpharmaceutically acceptable carrier suitable for administration of thedrug topically. The pharmaceutical compositions herein described mayalso comprise suitable solid of gel phase carriers or excipients.Examples of such carriers or excipients include, but are not limited to,calcium carbonate, calcium phosphate, various sugars, starches,cellulose derivatives, gelatin and polymers such as polyethyleneglycols.

The preparation of pharmaceutical compositions which contain peptides orpolypeptides as active ingredients is well known in the art. Typically,such compositions are prepared as indictable, either as liquid solutionsor suspensions, however, solid forms, which can be suspended orsolubilized prior to injection, can also be prepared. The preparationcan also be emulsified. The active therapeutic ingredient is mixed withinorganic and/or organic carriers, which are pharmaceutically acceptableand compatible with the active ingredient. Carriers are pharmaceuticallyacceptable excipients (vehicles) comprising more or less inertsubstances when added to a pharmaceutical composition to confer suitableconsistency or form to the composition. Suitable carriers are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents and pH buffering agents, which enhance theeffectiveness of the active ingredient.

Toxicity and therapeutic efficacy of the peptides described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the IC₅₀ (the concentrationwhich provides 50% inhibition) and the LD₅₀ (lethal dose causing deathin 50% of the tested animals) for a subject compound. The data obtainedfrom these cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition.

The amount of active agent used in an administration composition of thepresent invention is an amount effective to accomplish the purpose ofthe particular active agent for the target indication. The amount ofactive agent in the compositions typically is a pharmacologically,biologically, therapeutically, or chemically effective amount. However,the amount can be less than that amount when the composition is used ina dosage unit form because the dosage unit form may contain a pluralityof compounds or active agents in a single composition or may contain adivided pharmacologically, biologically, therapeutically, or chemicallyeffective amount. The total effective amount can then be administered incumulative units containing, in total, an effective amount of the activeagent.

A therapeutically effective amount of a peptide of the invention is anamount that when administered to a patient for treating aneurodegenerative disease, is capable of attenuating the progression ofsaid disease, alleviating symptoms of said disease and improvingmanagement of said disease.

Although an appropriate dosage of a peptide of the invention variesdepending on the administration route, age, body weight, sex orconditions of the patient, and should be determined by the physician inthe end, the dose suitable for adult humans can generally be betweenabout 0.2-2000 mg/kg body weight, about 0.2-1500 mg/kg body weight,about 0.2-1000 mg/kg body weight, about 0.2-500 mg/kg body weight, about0.2-200 mg/kg body weight, about 0.2-100 mg/kg body weight, about 1-2000mg/kg body weight, about 1-1500 mg/kg body weight, about 1-1000 mg/kgbody weight, about 1-500 mg/kg body weight, about 1-100 mg/kg bodyweight, or preferably between about 1-200 mg/kg.

The pharmaceutical compositions of the present invention comprise one ormore compounds of the present invention, and one or more excipients,carriers or diluents.

Carriers are pharmaceutically acceptable vehicles comprising more orless inert substances when added to a pharmaceutical composition toconfer suitable consistency or form to the composition. Suitablecarriers are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thecomposition can contain minor amounts of auxiliary substances such aswetting or emulsifying agents and pH buffering agents, which enhance theeffectiveness of the active ingredient.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

The compositions comprising the compounds and active agents have utilityin the delivery of active agents to selected biological systems and inan increased or improved bioavailability of the active agent compared toadministration of the active agent without the delivery agent. Deliverycan be improved by delivering more active agent over a period of time,or in delivering active agent in a particular time period (such as toeffect quicker or delayed delivery) or over a period of time (such assustained delivery).

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

The present invention further contemplates administration of othertherapeutic drugs to the subject. Exemplary drugs which may beadministered include, but are not limited to, oxidative agents,non-halogen activated-oxygen compounds, non-oxygen activated-halogencompounds, N-halo compounds and riluzole.

As mentioned above, in accordance with the present invention, the levelof Akt phosphorylation (pAkt) and pAkt:tAkt ratio may be assessed.

As used herein, the term “Akt” refers to the serine/threonine-specificprotein kinase also known as Protein Kinase B (PKB). Three genes in theAkt family, in humans: Akt1 (also called Akt), Akt2, and Akt3, encodefor enzymes that are members of this serine/threonine-specific proteinkinase family. Akt1 is involved in cellular survival pathways, byinhibiting apoptotic processes and is able to induce protein synthesispathways, which renders it a key signaling protein in the cellularpathways that lead to skeletal muscle hypertrophy, and general tissuegrowth. Since it can block apoptosis, and thereby promote cell survival,Akt1 has been implicated as a major factor in many types of cancer. Akt2is an important signaling molecule in the Insulin signaling pathway andis required to induce glucose transport. The role of Akt3 is less clear,though it appears to be predominantly expressed in brain.

As used herein, the term “pAkt” refers to the phosphorylated form ofAkt. According to exemplary embodiments, the Akt is phosphorylated onserine and/or theronine site(s), such as serine 308 and theronine 473.

As used herein, the term “pAkt:tAkt ratio” refers to the level ofphosphorylated Akt divided by the level of total Akt in the cell orbiological sample.

Phosphorylation is probably the most common of protein posttranslational modifications (PTMs), with 30% of eukaryotic proteinsestimated to be modified this way. Phosphorylation is essential to thecell by playing a central role in signal transduction cascades,regulation of protein activity and protein-protein interactions. Proteinphosphorylation can be detected as a mass shift (+79.99 Da) in massspectra, which corresponds to the addition of HPO₃ to a peptide,generally at serine, threonine or tyrosine residues.

Methods for measuring protein and peptide phosphorylation (i.e. pAkt)are known in the art and include Kinase Activity Assays, Western blots(i.e. with an anti-pAkt antibody) or Enzyme-Linked Immunosorbent Assays(ELISA, i.e. with an anti-pAkt antibody) and Mass Spectrometry (e.g.Donahue et al., Methods in Enzymology, Volume 434, p. 131-150).Similarly, total Akt (tAkt) levels may be assed using e.g. Western blot(i.e. with an anti-Akt antibody) or Enzyme-Linked Immunosorbent Assay(ELISA i.e. with an anti-Akt antibody).

Without being bound by any theory or mechanism, Akt pathway dysfunctionin ALS patients may result from multiple effects of the underlying ALSdisease process. Akt is the major signaling pathway activated by atleast four major factors and hormones: GH, IGF1, HGF and VEGF. Aktpathway induction by all four is impaired in ALS:

-   -   i. GH levels are drastically reduced as a direct result of        oxidative stress and other primary disease processes of ALS.    -   ii. The IGF1 signaling pathway, a major fallback option of GH,        is disturbed by up-regulation of IGFBP1 and direct carbonylation        damage to the IGF1 Receptor.    -   iii. Later in the disease, IGF1, HGF and VEGF levels are also        decreased, further reducing the natural stress response of the        neuro-musculature system.

As shown in Examples 4 and 5 of the Examples section which follows, thepresent inventors have shown that pAkt correlates with the state of thedisease, i.e. in rapidly progressing ALS disease the ratio ofmuscle/lymph pAkt is significantly lower compared to slow progressingALS disease. Moreover, the present inventors have shown that pAkt can beused to assess responsiveness to treatment, i.e. when the subjectresponds to treatment, there is a significant elevation in pAkt levels.

Assessing the level of pAkt or pAkt:tAkt ratio may thus be carried outprior to treatment, upon treatment and following treatment and may beused for various applications as specified in further detail below.

According to one embodiment, there is provided a method of assessingresponsiveness to treatment of a disease (e.g. ALS) with the peptide ofthe present invention, the method comprising: assessing the level ofpAkt and pAkt:tAkt ratio in bodily sample, such as, a bodily samplederived from the muscle, the blood or lymphatic system of the subject(e.g. comprising lymphocytes), wherein responsiveness to treatment isindicated by a pAkt level or pAkt:tAkt ratio which is significantlyabove a value of pAkt level and pAkt:tAkt ratio in the subject prior tothe treatment.

As used herein, the terms “above” or “increase” as used herein, refer toat least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90% or at least about 100% higherlevel of pAkt level or pAkt:tAkt ratio in the subject followingtreatment as compared to a level of same in a subject prior to treatmentor to commonly used control samples taken from sick individual (e.g. anindividual with a disease, e.g. ALS, corresponding to the subject beingtreated).

In another aspect, the present invention includes a method fordetermining the efficacy of treatment for a neurodegenerative disease ina subject in need thereof, the method comprises determining in a samplefrom the subject whether a composition comprising a therapeuticallyeffective amount of a peptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and an analog ora derivative thereof induces an increase in the level of pAkt andpAkt:tAkt ratio. According to the principles of the present invention,an increase in the level of pAkt and pAkt:tAkt ratio indicates that thesubject will show responsiveness to treatment with said peptide.

In some embodiments, the level of pAkt:tAkt ratio (also denoted“pAkt/tAkt” standing for phosphorylated Akt divided by total Akt) insaid bodily sample is evaluated, wherein responsiveness to treatment isdetermined for a subject having a pAkt/tAkt ratio which is significantlylower than a corresponding control value.

As used herein, the terms “below” or “lower” as used herein, refer to atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90% or at least about 100% lower levelof pAkt level or pAkt:tAkt ratio in the subject following treatment ascompared to a level of same in a control sample (e.g. a sample takenfrom healthy individual).

Preferably, the control value corresponds to pAkt level or pAkt/Aktratio in a sample derived from a healthy individual, or from a panel ofcontrol samples obtained from a set of healthy individuals, or from astored set of data corresponding to control individuals (e.g. healthyindividuals or individuals that are not afflicted with neurodegenerativedisease, particularly ALS).

According to yet another embodiment, the control value corresponds topAkt level in a sample derived from a bodily sample of an individualafflicted with a neurodegenerative disease, e.g. ALS, but manifesting amilder version of the disease according to measureable criteria (e.g.slowly progressing disease).

In the methods of the invention, the terminology of “significantreduction”, “significant decline”, “significantly below” and the like,in the level or amount of pAkt of an individual, is interchangeable andrefers to a statistically significant reduction recognized by a skilledartisan, as compared to control. Statistical significance may beevaluated by any method known in the art, such as, Student's T-test,Analysis of variance (ANOVA) and Chi-square test, among others.

The present invention also highlights the use of Akt phosphorylation asa biomarker. Specifically, the present invention provides a method forassessing whether the progression of ALS is slow or rapid.

By “rapid progression” or “rapid disease” it is meant that the symptomsof ALS progress continuously and significant degradation of motorneurons can be observed within less than a year. Rapid disease in humanpatients corresponds to survival of up to 4 years from diagnosis. Incontrast, “slow progression” or “slow disease” refers to a conditionwhere the ALS symptoms appear slowly with long periods, i.e. many monthsor years. Slow disease in human patients also typically corresponds tosurvival of more than 4 years from diagnosis.

According to one embodiment, there is provided a method forprognosticating the progression of amyotrophic lateral sclerosis (ALS)in an ALS patient, comprising (a) assessing the value of at least onemarker selected from: pAkt and pAkt:tAkt ratio, in a bodily samplederived from the ALS patient; and (b) obtaining the ratio between thevalue of said marker and the value of the marker in a control sample,wherein a level of pAkt or pAkt:tAkt ratio significantly below a controlvalue indicates a rapid disease.

The present invention further provides kits suitable for use in methodsof the invention. Specifically, the present invention provides kits fordiagnosing neurodegenerative diseases and kits for diagnosing ALS. Inaddition, the present invention provides kits for prognosticating theprogression of neurodegenerative diseases and kits for prognosticatingthe progression of ALS in an ALS patient.

A kit according to the present invention preferably comprises i) meansfor collecting a bodily sample from a subject ii) means for determiningthe level of the markers pAkt and tAkt (total Akt) in the sample andiii) a standard sample for comparison of the tested sample.

In certain embodiments, the means for determining the levels of saidmarkers comprise at least one antibody directed to pAkt and at least oneantibody directed to total Akt. In some embodiments, the level of pAktand tAkt are determined by Western blot analysis, or applying Aktphosphorylation assays as been demonstrated by Armentero et al.(Neurobiol Aging, Jan. 25, 2010). In other embodiments, the level ofpAkt and tAkt are determined by FACS (fluorescence-activated cellsorting) assays.

According to one embodiment, the kits further comprise a standard, acalibration curve or an index indicating a control value of the pAkt andthe tAkt.

Typically a control value corresponds to pAkt level or pAkt:tAkt ratioin a sample comprising a bodily sample of a healthy individual, a bodilysample of an individual not afflicted with any neurodegenerativedisease, such as ALS, a bodily sample of an individual afflicted with aslowly progressing neurodegenerative disease, such as ALS.

The kits for prognosticating the progression of ALS, may preferablycomprise (a) means for determining pAkt levels and tAkt level in abodily sample, such as, a bodily sample derived from the peripheral orlymphatic systems, (b) means for determining pAkt levels in and tAktlevel in a bodily sample derived from the muscles of a subject and iii)a standard sample for comparison of the tested sample.

In certain embodiments, the detection of pAkt and tAkt may be performedusing an immunoassay such as an enzyme-linked immunosorbent assay(ELISA) testing kit. In such assays samples are typically incubated inthe presence of an immobilized first specific binding agent (e.g. anantibody) capable of specifically binding pAkt or an immobilized firstspecific binding agent capable of binding tAkt. Binding of pAkt or tAktto said first specific binding agent may be measured using any one of avariety of known methods, such as using a labeled second specificbinding agent capable of specifically binding pAkt or tAkt,respectively, (at a different epitope) or capable of specificallybinding the first specific binding agent.

Exemplary specific binding agents include e.g. monoclonal antibodies,polyclonal antibodies, and antibody fragments such as recombinantantibody fragments, single-chain antibodies (scFv) and the like.

Exemplary anti-pAkt antibodies which may be used in accordance with thepresent teachings may be commercially purchased from e.g. Cell SignalingTechnology (e.g. #9275), Millipore (e.g. STAR phospho-Akt1 (Thr308)ELISA kit), BioLegend and Epitomics Inc.

Exemplary anti-tAkt antibodies (i.e. anti-Akt antibodies) which may beused in accordance with the present teachings may be commerciallypurchased from e.g. Cell Signaling Technology (e.g. #9272), Millipore(e.g. STAR Akt ELISA kit), Enzo Life Sciences Inc., BioLegend andProteintech Group Inc.

In some embodiments, various conventional tags or labels may be used,such as a radioisotope, an enzyme, a chromophore or a fluorophore. Atypical radioisotope is iodine-125 or sulfur-35. Typical enzymes forthis purpose include horseradish peroxidase, horseradish galactosidaseand alkaline phosphatase.

Alternately, other immunoassays may be used; such techniques are wellknown to the ordinarily skilled artisan and have been described in manystandard immunology manuals and texts.

In some embodiments, the methods of the invention are suitable forautomated or semi-automated analysis, and may enable clinical, medium orhigh-throughput screening of multiple samples. For example, automatedELISA systems such as Biotest's Quickstep® ELISA Processor, MaxmatAutomated microwell ELISA analyzer (Maxmat S. A., France), or DSX™Four-Plate System (Dynex Technologies) may conveniently be used.

Other suitable assays include for example flow cytometry assays (such assingleplex and multiplex bead-based Luminex® assays (Invitrogen).

Alternately, pAkt or tAkt may be captured on an antibody microarray. Theantibody microarray comprises an anti-pAkt antibody or an anti-tAktantibody, or, for example, a combination of anti-pAkt antibodies andanti-tAkt antibodies. In general, the sample (e.g., peripheral blood)obtained from the subject is placed on the active surface of a chip fora sufficient time to allow binding. Then, unbound molecules are washedfrom the surface using a suitable eluant, such as phosphate bufferedsaline. In general, the more stringent the eluant, the more tightly pAktor tAkt must be bound to be retained after the wash. As a result, theretained pAkt or tAkt can be detected by appropriate means.

Additional exemplary assays may be based on dipstick technology, asdemonstrated, for example, in U.S. Pat. Nos. 4,632,901; 4,313,734;4,786,589 5,656,448 and EP 0125118. For example, U.S. Pat. No.4,632,901, discloses a flow-through type immunoassay device comprisingantibody (specific to a target antigen analyte) bound to a porousmembrane or filter to which is added a liquid sample. As the liquidflows through the membrane, target analyte binds to the antibody. Theaddition of sample is followed by addition of labeled antibody. Thevisual detection of labeled antibody provides an indication of thepresence of target antigen analyte in the sample. EP 0125118 discloses asandwich type dipstick immunoassay in which immunochemical componentssuch as antibodies are bound to a solid phase. The assay device is“dipped” for incubation into a sample suspected of containing unknownantigen analyte. Enzyme-labeled antibody is then added, eithersimultaneously or after an incubation period. The device next is washedand then inserted into a second solution containing a substrate for theenzyme. The enzyme-label, if present, interacts with the substrate,causing the formation of colored products which either deposit as aprecipitate onto the solid phase or produce a visible color change inthe substrate solution.

For example, the method may be performed by the steps comprising:

a) collecting a bodily sample from the subject;

b) contacting the sample, under conditions such that a specificantigen-antibody complex may be formed, with at least one antibody, saidantibody being directed to either pAkt or tAkt;

c) quantifying the amount of antigen-antibody complex formed, whereinsaid amount is indicative of the amount of pAkt or tAkt in said sample.

An antibody “directed to” an antigen, as used herein is an antibodywhich is capable of specifically binding the antigen. The term“specifically bind” as used herein means that the binding of an antibodyto an antigen is not competitively inhibited by the presence ofnon-related molecules. Antibodies directed to pAkt and antibodiesdirected to tAkt may be prepared using well known methods, for exampleas detailed hereinabove. Alternatively, antibodies, or ELISA kits fordetermining the presence of these antigens, may be purchased from avariety of sources.

The following examples are to be considered merely as illustrative andnon-limiting in nature. It will be apparent to one skilled in the art towhich the present invention pertains that many modifications,permutations, and variations may be made without departing from thescope of the invention.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 The Effect of the Peptide of SEQ ID NO: 1 on the Akt Pathwayin Cells

Raw264.7 macrophages were treated with 50 mM of the peptide having theamino acid sequence of SEQ ID NO: 1 (10×⁵ per well) in quadruplicates.Akt phosphorylation was found to be induced shortly after introductionof the peptide (FIG. 1A). Akt and pAkt were detected using western blotanalysis with Cell Signaling Technology #9272 and #9275 antibodies. Thisobservation demonstrates that the peptide triggers activation of the Aktpathway in a specific manner.

Next, mice were injected with the peptide of SEQ ID NO: 1 (400 μl) andafter 30 minutes lymph nodes were assessed for pAkt, compared to lymphsamples obtained from non-treated mice. Akt phosphorylation was measuredusing ELISA compared to a standard, using Millipore's STAR Akt and STARphospho-Akt1 (Thr308) ELISA kits.

As shown in FIG. 1B, Akt phosphorylation was higher in treated micecompared to non-treated mice.

Example 2 The Effect of the Peptide of SEQ ID NO: 1 on Survival of aMice Model for ALS

ALS is characterized by apoptosis of motor neurons. The SOD1G93A mousemodel of familial ALS was used in the following examples. Initially,doses less than optimal were applied, and model irregularities due togene counts were detected. These deficiencies were repaired in laterstudies.

Mice were purchased from Jackson and were administered with a dose of atleast 200 microgram a day of the peptide of SEQ ID NO: 1. Overall, thestudies included 142 mice. Eventually all mice died of neurologicdisease. Mice (a few) that died before the detection of a symptomaticdisease were not included, as recommended by Scott et al. (AmyotrophLateral Scler. 2008; 9(1):4-15). Of the 142 mice, 99 mice were treatedintraperitoneally (IP), intranasally (IN), intravenously (IV) or with acombined delivery, and 43 mice were used as control (untreated ortreated with PBS).

Allocation of the mice to the experimental groups was done by matchingweight and, when applicable (particularly in study III) also by gendermatch. In studies II and III, where treatment started after diseaseonset, the primary grouping criteria was date of onset. In fact, age atonset and weight at onset reflect to a certain degree the diseaseseverity, thereby allowing to perform an even distribution of animalsaccording to the phenotypic manifestation of the disease. Althoughgenotypic distribution and separation of siblings is recommended,siblings in the same groups were used, preferring the phenotypiccriteria as an indicator of the severity of the disease itself, and asin many cases too many of the mice were from the same pedigree.

Exclusion (censoring) criteria were employed to exclude data that do notinclude any measurements taken from animals that died before detectionof the neurological damage or animals that died due to non-diseaseassociated reasons (such as physical wounds).

In this example, 40 female mice (purchased from Jackson) were included.30 mice were treated (IN or IV; peptide of SEQ ID NO: 1) and 10 micewere controls. Disease onset was measured by Rotarod exertion value(measuring mobility on a Rotarod vs. time), defined as Rotarod valuesbelow 180 (i.e. 3 min.). The animals were divided into groups accordingto weight match.

TABLE 1 Study design Age Group Treatment Route Schedule at treatmentinitiation n 1 PBS (control) IV Daily 75 days 10 2 200 μg peptide IVDaily 75 days 10 3 400 μg peptide IV Daily 75 days 10 4 400 μg peptideIN Daily 75 days 10

TABLE 2 Total survival of mice treated with the peptide Added survivalSurvival of Survival compared to 50% Mean control Group (days) (days) P(Logrank) (days) Control 125 128.1 — 400 IN 139 132.4  4-14 200 IV 145144.3 0.02 16-20 400 IV 139 136.2  8-14 All 139 139.2 0.017* 11-14treatment groups *Significance equivalent to p = 0.05 for 3 combinedgroups

The results summarized in Table 2 above clearly indicate that thepeptide of the invention induces a significant (p=0.05) increase insurvival, additional 11 to 14 days as compared to control, irrespectiveof the disease stage. The significantly prolonged survival induced bythe peptide of the invention is also shown in FIG. 2.

Similar results are shown in the ‘all stages’ columns of Table 3, below.As detailed in Table 3, the most pronounced effect of the protein of theinvention is exerted during late stages of the disease, which ischaracterized by low Rotarod index (less than 120).

TABLE 3 Disease duration at different stages of the disease (late andearly) upon treatment with the peptide of the invention at 200 μg or 400μg for 7 successive days^(a) Late disease Early disease All stages ofthe (Y < 120)^(a) (180 > Y > 120)^(b) disease Duration Duration DurationGroup (days) (days) (days) 400 μg IN 26.9 17.7 44.6 (n = 10) 200 μg IV34.8 16.2 51.0 (n = 10) 400 μg IV 25.7 15.8 41.5 (n = 10) All 29.2 16.645.7 treatments (n = 30) Control 15.5 15.7 31.2 (10) ^(a)Mice stayed onthe Rotarod for less than 120 seconds, i.e. Rotarod index (Y) < 120.^(b)Mice that stayed on the Rotarod for more than 120 seconds but lessthan 180 seconds.

The results indicate that daily treatments with the peptide of theinvention via any way of administration (IN or IV) leads to increasedsurvival at advanced (late) stages of the disease (measured by Rotarodexertion values below 120 seconds). Treatment with the peptide of theinvention prolonged the duration at the late stage by 88% while notaffecting disease onset or disease progression at the earlier stages. Inthis study, onset in all groups was at an average age of 93-97 days, andthere were no differences between the groups. Late-stage disease startedat age of about 110 days.

In another study (FIG. 2) the differences between 21 mice treated with400 microgram (200 IP+200 IV) of the SEQ ID NO: 1 peptide and 21non-treated mice are observed in weeks 18-20. At week 18, 21/21 treatedvs. 16/21 non-treated manifested at 20% difference in survival (*,P=0.02); at week 19, 19/21 treated vs. 9/21 non-treated manifested 50%difference in survival (***, P=0.001); and at week 20, 14/21 treated vs.6/21 non-treated manifested a 40% difference in survival (**, P=0.005).

The following analysis (Table 4, below) shows the effect of the peptideof the invention on all treatments, irrespective of the stage ofdisease, but subject to the method of administration.

TABLE 4 Summary of 50% survival in all groups (treatments and control)No. of No. of Age at Treatment Control Average ± Average treatmentanimals animals SD of added added initiation (number (number of survivalSignificance 50% Study Treatment (days) of groups) groups) (days)(Logrank) survival Total 200-600 μg 89.75 99 (9) 43 (3) *7.2 ± 12.60.008 10 IP/IV/IN §Disease onset was observed *Adjusted according tocontrol values

The data shows that the 50% survival of the 99 mice treated with thepeptide of SEQ ID NO: 1 was about 10 days longer, significantly(P=0.008) as compared to control. The most effective route ofadministration seems to be a combined IV/IP administration.

Example 3 The Effect of the Peptide of the Invention on DiseaseProgression

The protective effect of the peptide of the invention is not onlylimited to prolonged survival, but also involves significant delay inthe progression of disease symptoms, as monitored by the neurologicdisability score, thereby postponing development of end-stage disease.

Mice were administered with the peptide of SEQ ID NO: 1 or placebo (PBS)starting at disease onset as measured by weight loss. It was at averageage of about 90 days. The mice were treated daily. During the period ofweeks 16-19, the neurologic score was assessed, and was found lower by afactor of about 2 in treated mice compared to controls (FIG. 3).

Example 4 The Effect of the Peptide of SEQ ID NO: 1 on the Akt PathwayIn Vivo

Twenty SOD1 mice at the age of 75 days (i.e. prior to disease onset)were treated daily with 200 μIV+200 μg IP of the peptide of SEQ ID NO: 1or with PBS, and monitored for body weight and neurological score. Atthe age of 120 days, the mice were sacrificed, and phosphorylated Akt(pAkt) and total Akt (tAkt) in lymphocytes (lymph node) and muscle cells(hind legs) were measured (see Tables 5-6, below). Two treated mice diedprior to age 120 days without any preceding signs of neurologic damage.Two non-treated mice died prior to age 120 days after detection ofsevere signs of neurologic disease.

TABLE 5 The effect of the peptide of SEQ ID NO: 1 on Akt phosphorylationMuscles Lymph node pAkt pAkt/tAkt pAkt pAkt/tAkt Control (n = 8) 1501.61  95 0.96 SEQ ID NO: 1 149 1.83 129 1.35 (n = 8) Difference −1% +14%+36% +42%

TABLE 6 Treatment with the peptide of SEQ ID NO: 1 significantlyincreases the combined pAkt/Akt ratio of lymph nodes and muscles MuscleAverage Percentage of Lymph node pAKT Average pAKT mice with highTreatment pAKT ratio ratio pAKT ratio pAkt content PBS 0.96 1.61 1211.27 12.5% SEQ ID 1.35 1.83 140 1.61 62.5% NO: 1 Percent 42% 14% 16% 26%change P value 0.11 0.45 0.037 0.07 0.04 chi square

The peptide of SEQ ID NO: 1 increased the phosphorylation level oflymphocytes by 36%, and even more in terms of the pAkt/tAkt ratio (42%).There was a minimal effect on muscle cells, which were predominantlyhighly phosphorylated (70% higher pAkt/tAkt ratio in muscles compared tolymphocytes, before treatment with the peptide of the invention). Theaverage increase of pAkt in both muscles and lymphocytes wasstatistically significant—P=0.04.

Preliminary measurements of weight loss and neurologic score amongnon-treated mice with naturally elevated peripheral (muscle and lymph)pAkt showed differences in clinical parameters, as detailed in Table 7,below. This table demonstrates that low lymphocyte and muscle pAktcorresponds to rapid disease (characterized by high neurologic score andweight loss at 120 days) wherein relatively high lymphocyte pAkt andmuscle pAkt corresponds to slow disease (i.e. a disease characterized bylow neurologic score and weight loss at 120 days).

It is to be understood that a combination of weight loss andneurological score is considered one of the most reliable and gentle(i.e. not introducing additional stressors) ways to assess the presence,progression and staging of ALS.

TABLE 7 The effect of Akt phosphorylation on disease parameters innon-treated SOD1 mice aged 120 days. Muscle- Previous Muscle Lymphocytelymph Neurologic week pAkt pAkt pAkt score weight loss Lower 138  94 1160.67 4.7% peripheral pAkt (3) Higher 151 111 131 0.33 1.4% peripheralpAkt (3) Change +9% +18% +13% −50% −71% 

The correlation of Akt phosphorylation with disease progression is evenmore striking when peripheral (muscle and lymph) pAkt of mice with rapiddisease, both treated and non-treated, is compared to peripheral pAkt ofmice with slow disease, in both treated and non-treated SOD1 mice, asdescribed in Table 8 below.

TABLE 8 The effect of Akt phosphorylation on rapid and slow diseaseNeurologic Muscle + lymph score pAkt Score 0.5-1.25 (n = 7, SOD1 micewith 0.8 120 Rapid Disease) Score 0-0.25 (n = 9, SOD1 mice with 0.05 139Slow Disease) Significance P = 0.01

In this study, animals at day 120 were divided into groups by thedisease state. The results of this analysis indicate that the pAKTcorrelates with the state of the disease.

Specifically, in mice where the disease progressed rapidly (neurologicalscore of 0.8 at 120 days), the ratio of muscle:lymph pAkt issignificantly (P=0.01) lower than this ratio in mice where the diseaseprogressed slowly (neurological score of 0.05 at 120 days). Thus, theresults indicate that pAkt phosphorylation is a reliable indication fordistinguishing the stage of the ALS disease (slow/fast).

When these mice were divided according to pAkt ratio of lymph node andmuscle, 10 mice with low pAkt had a significantly (P=0.005) 10 timeshigher clinical score (0.6) as compared to 6 mice with high pAkt (0.06).

Thus, the peptide of the invention might employ increased pAkt in bothmuscles and lymphocytes for reduction of ALS progression rate in SOD1mice, and might contribute to the treatment of sporadic ALS.

Example 5 Responsiveness to Treatment

The following analysis reflects the significant therapeutic effect ofthe peptide of the invention on ALS and emphasizes the importance inassessing responsiveness to treatment in terms of increased biomarkerduring the treatment.

Mice, age 75 days, were treated for 45 days with 400 μg of the peptideof SEQ ID NO: 1, administered as follows: 200 μg IV and 200 μg IP. Atage of 120 days, 6 of the peptide treated mice responded to treatment,having a slow disease e.g. low weight loss (only 3.3%) and lowneurological score (0.08) compared to placebo (PBS) treated mice whichkept losing weight, reaching a weight loss of 5.4% and neurologicalscore of 0.8. The six responding mice had 49% higher pAkt (P=0.01)compared to the placebo treated mice.

Two other mice treated with the peptide of the invention did not respondto treatment, having a significantly higher neurological score of 1.1(93%; P=0.05) and higher weight loss (6.3%). Those mice resulted with asignificantly lower pAkt phosphorylation (−37%; P=0.004 in comparisonwith the responding group).

The data is summarized in Table 9, below.

TABLE 9 pAkt in responding and non-responding SOD1 mice at 120 daysAverage score (age Lymphocyte Effect on 110-120 Effect on pAkt pAkt*days) score** Weight loss^(§) Placebo 95 100% 0.8 100% 5.4% (PBS; n =10) (8 survivors, 2 died earlier) Responding 142  +49% pAkt* 0.08 −90%score* 3.3% (SEQ ID P = 0.01 P = 0.02 NO: 1; n = 6) Non 90 −37% pAkt**1.1 +1400%** 6.3% responding P = 0.004 score (SEQ ID P = 0.05 NO: 1; n =2) *The effect of treatment on pAkt and neurologic score relative toplacebo (100%). **The effect of treatment on pAkt and neurologic scorein non-responders relative to responders ^(§)Average weight loss(compared to the weight on the week prior to treatment)

Example 6 Measuring the Effect of the Peptide of SEQ ID NO: 1 on Slowand Rapid Disease

SOD1 mice were treated IP and IV using the peptide of SEQ ID NO: 1starting at disease onset, detected by weight loss, at an average age of90 days.

In order to check if mice with rapid disease respond to treatmentdifferently from mice with slow disease, a comparison was madeseparately of the 50% control and treated mice that survived less thanor equal to the median survival and 50% control and treated mice thatsurvived longer than the median survival.

According to this comparison, demonstrated in Table 10 below, SD valueswere 40% lower for treated mice, demonstrating that treatment reducedthe diversity and range between rapid and slow disease, making the rapiddisease look more like slow disease, and the extension of survival wasmuch larger (11 days comparing to 3 days), and much more significant(P=0.0001 compared to P=0.8), for mice with fast disease.

TABLE 10 Systemic treatment with the peptide of SEQ ID NO: 1 starting atthe onset of weight loss (age 90 days) N SD 50% fast 50% slow control/control/ control/ control/ treatment treatment treatment treatment PBS21 13.8 125.2 146.7 SEQ ID NO: 1 21 8.7 136.0 149.7 Significance P =0.0001 P = 0.8

The absence of an effect on long-term survivors was also reflected bythe survival and disease progression curves: they showed the convergenceof control and treatment curves for long-term surviving mice and at latestudy stages, which demonstrate the lack of effect on the fraction of25% of mice with the longest survival (FIGS. 2 and 3).

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting. In addition, any priority document(s) of this applicationis/are hereby incorporated herein by reference in its/their entirety.

What is claimed is:
 1. A method for treating amyotrophic lateralsclerosis (ALS) in a subject in need thereof, comprising administeringto a subject having a rapidly progressing ALS a therapeuticallyeffective amount of an agent capable of activating a Akt pathway,thereby treating the ALS.
 2. The method according to claim 1, whereinsaid agent capable of activating said Akt pathway comprises a peptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2 and an analog or a derivative thereof.
 3. Themethod according to claim 1, wherein said agent capable of activatingsaid Akt pathway is selected from the group consisting of a insulin-likegrowth factor 1 (IGF-I), vascular endothelial growth factor (VEGF),angiogenin, naphtho[1,2-b]furan-4,5-dione (NFD), ciliary neurotrophicfactor (CNTF), brain-derived neurotrophic factor (BDNF), growthfactor-beta (TGF-beta), glial-cell-line-derived neurotrophic factor(GDNF), Hepatic growth factor (HGF), fibroblast growth factor (FGF) andepidermal growth factor (EGF).
 4. A method for treating amyotrophiclateral sclerosis (ALS) in a subject in need thereof, comprising (a)assessing the level of pAkt and pAkt:tAkt ratio in bodily sample derivedfrom the subject; and (b) administering a therapeutically effectiveamount of a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2 and an analog or aderivative thereof, to a subject having pAkt level or pAkt:tAkt ratiosignificantly below a control value.
 5. The method according to claim 4,wherein the bodily sample is derived from the peripheral blood, thelymph system or the muscle of the subject.
 6. The method according toclaim 5, wherein said peripheral blood or said lymph system compriseslymphocytes.
 7. The method according to claim 4, wherein the controlvalue corresponds to pAkt level or pAkt:tAkt ratio in a sample selectedfrom the group consisting of: a bodily sample of a healthy individual, abodily sample of an individual not afflicted with ALS, and a samplederived from a ALS subject having a slow disease.
 8. The methodaccording to claim 4, wherein the peptide is comprised in apharmaceutical composition in combination with at least one moretherapeutic drug.
 9. The method according to claim 8, wherein the atleast one more therapeutic drug is selected from the group consistingof: an oxidative agent, non-halogen activated-oxygen compounds,non-oxygen activated-halogen compounds, N-halo compounds and riluzole.10. The method according to claim 8, wherein the pharmaceuticalcomposition further comprises a pharmaceutical acceptable excipient,carrier or diluent.
 11. The method according to claim 4, whereintreating comprises attenuating the progression of said ALS, alleviatingsymptoms of said ALS, delaying the appearance of ALS symptoms orimproving management of said ALS.
 12. The method according to claim 4,wherein treating comprises administering the peptide by a route ofadministration selected from the group consisting of: oral, transdermal,parenteral, transmucosal, intrathecal, intracerebroventricular (ICV),intranasal, sublingual, intravenous and intraperitoneal.
 13. The methodaccording to claim 4, wherein the peptide consists of an amino acidsequence selected from SEQ ID NO: 1, SEQ ID NO: 2 and an analog or aderivative thereof.
 14. A method for assessing responsiveness totreatment of a ALS with a peptide comprising an amino acid sequence ofSEQ ID NO: 1, SEQ ID NO: 2 or an analog or a derivative thereof, or apharmaceutical composition comprising same, the method comprising:treating the subject with the peptide; and assessing the level of pAktor pAkt:tAkt ratio in bodily sample derived from a subject, whereinresponsiveness to treatment is indicated by a pAkt level or pAkt:tAktratio significantly above a value of a pAkt level or pAkt:tAkt ratio insaid subject prior to said treatment.
 15. The method according to claim14, wherein the bodily sample is derived from the peripheral blood, thelymph system or a muscle of said subject.
 16. The method according toclaim 15, wherein said peripheral blood or said lymph system compriseslymphocytes.
 17. The method according to claim 14, wherein the controlvalue corresponds to pAkt level or pAkt:tAkt ratio in a sample selectedfrom the group consisting of: a bodily sample of a healthy individual, abodily sample of an individual not afflicted with any neurodegenerativedisease and a sample derived from a ALS subject having a slow disease.